Brain Research Institute, Medical Faculty of the University of Zürich and Department of Biology of the Swiss Federal Institute of Technology Zürich, Switzerland.
Institute of Pharmacology and Toxicology, Medical Faculty of the University of Zürich Zürich, Switzerland.
Front Cell Neurosci. 2014 Mar 19;8:62. doi: 10.3389/fncel.2014.00062. eCollection 2014.
Gene knockout by homologous recombination is a popular method to study gene functions in the mouse in vivo. However, its lack of temporal control has limited the interpretation of knockout studies because the complete elimination of a gene product often alters developmental processes, and can induce severe malformations or lethality. Conditional gene knockdown has emerged as a compelling alternative to gene knockout, an approach well-established in vitro but that remains challenging in vivo, especially in the adult brain. Here, we report a method for conditional and cell-specific gene knockdown in the mouse brain in vivo that combines Cre-mediated RNA interference (RNAi) with classical and lentivirus-mediated transgenesis. The method is based on the inducible expression of a silencing short hairpin RNA (shRNA) introduced in mice by lentivirus-mediated transgenesis, and on its activation by excision of a floxed stop EGFP reporter with an inducible Cre recombinase expressed in astrocytes or in neurons. This dual system should be of broad utility for comparative studies of gene functions in these two cell types in vivo.
基因敲除通过同源重组是一种流行的方法来研究在体内的小鼠基因功能。然而，其缺乏时间控制已经限制了敲除研究的解释，因为一个基因产物的完全消除通常会改变发育过程，并能引起严重的畸形或致死。条件性基因敲低已经成为基因敲除的一个有吸引力的替代方法，该方法在体外已经建立，但在体内，特别是在成年大脑中仍然具有挑战性。在这里，我们报告了一种在体内的小鼠大脑中进行条件性和细胞特异性基因敲低的方法，该方法将 Cre 介导的 RNA 干扰（RNAi）与经典和慢病毒介导的转基因技术相结合。该方法基于诱导表达沉默短发夹 RNA（shRNA），通过慢病毒介导的转基因技术引入小鼠，并用在星形胶质细胞或神经元中表达的诱导型 Cre 重组酶切除 floxed 停止 EGFP 报告基因进行激活。这个双重系统应该对这两种细胞类型的基因功能进行比较研究具有广泛的应用。
